My PI and I are currently working with multiple melanoma cell lines to determine the effectiveness of treatment. However, we have identified that there maybe some issues with the primers resulting in serious primer dimer, and even non-specific amplification resulting in some competing secondary products. I've added some of the data including some melt peak analysis that explain the issue. Does anyone have a solid set of primers for GAPDH, CXCL1, CD26 that work well in melanoma cells with no primer dimer or nonspecific amplification? Please let me know, and thanks for everyone support and suggestions!
Additional Details: We are currently performing RT-PCR separate from qPCR reactions so we don't need hotstart, and loading 1µL of primers at 10µM for 10µL reaction.
Would you consider using a different housekeeping gene besides GAPDH? Not that there's anything wrong with using it, but I personally have had success with using B-actin primers, so I could provide sequences for those if you want to try something else.
That is a great suggestion! I'll check with my PI and get back to you! Thanks again for reaching out! I really do appreciate it.
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